Throughout this application various publications are referred to by partial citations within parenthesis. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications, in their entireties, are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
In vertebrate embryos, the neural tube displays distinct cell types at defined dorsoventral positions. Floor plate cells differentiate at the ventral midline; motor neurons appear in ventrolateral regions; and sensory relay neurons, neural crest, and roof plate cells appear dorsally. The generation of cell pattern in the neural tube depends on signals that derive from surrounding tissues. A clear example of this is the influence of axial mesoderm on the development of ventral cell types.
The differentiation of floor plate cells, motor neurons, and other ventral cell types requires inductive signals from axial mesodermal cells of the notochord. In the absence of the notochord, floor plate cells and motor neurons do not differentiate (Placzek et al., 1990b; Bovolenta and Dodd, 1991; Clarke et al., 1991; van Straaten and Hekking, 1991; Yamada et al., 1991; Ruiz l Altaba, 1992; Goulding et al., 1993; Ruiz l Altaba et al., 1993a; Halpern et al., 1993). Conversely, notochord grafts can induce the ectopic differentiation of floor plate cells and motor neurons in vivo and in vitro (van Straaten et al., 1988; Placzek et al., 1990b, 1991, 1993, Yamada et al., 1991, 1993; Ruiz l Altaba, 1992; Goulding et al., 1993). Floor plate cells themselves also possess both floor plate and motor neuron inducing activity (Yamada et al., 1991, 1993; Hatta et al., 1991; Placzek et al., 1993). In vitro assays have provided evidence that floor plate induction requires a contact-mediated signal, whereas motor neurons can be induced by diffusible signals (Yamada et al., 1993; Placzek et al., 1990b, 1993).
The differentiation of floor plate cells and motor neurons is associated with the expression of different classes of transcription factors. Floor plate cells express three members of the hepatocyte nuclear factor HNF-3/fork head gene family (Weigel and Jackie, 1990, Lai et al., 1991):Pintallavis (XFKH1/XFD1/1), HNF-3.beta., and HNF-3a (Dirksen and Jamrich, 1992; Knochel et al., 1992; Ruiz l Altaba and Jessell, 1992; Bolce et al., 1993; Monaghan et al., 1993; Ruiz l Altaba et al., 1993a; Sasaki and Hogan, 1993; Strahle e al., 1993). Ectopic expression of Pintallavis and HNF-3.beta. leads to the appearance of floor plate markers in cells in the dorsal region of the neural tube (Ruiz l Altaba et al., 1992, 1993b; A. R. A. et al., unpublished data; Sasaki and Hogan, 1994), suggesting that members of this family may specify floor plate cell fate. The differentiation of motor neurons is associated with expression of islet-1, a member of the LIM homeobox gene family (Ericson et al., 1992; Yamada et al., 1993). In addition to their possible functions in cell fate determination, these transcription factors provide markers that can be used in conjunction with cell surface molecules to monitor floor plate and motor neuron differentiation.
Cell patterning in the dorsal neural tube appears to be regulated by members of two families of secreted proteins that also have prominent roles in insect development. The transforming growth factor .beta. (TGF.beta.) family member dorsalin-1 is expressed in the dorsal neural tube and can induce the differentiation of neural crest cells in neural plate explants in vitro (Basler et al., 1993). Members of the wnt family are also expressed in the dorsal neural tube (Roelink and Nusse, 1991; Nusse and Varmus, 1992; Parr et al., 1993). In Drosophila, the TGF.beta. family member decapentaplegic. (dpp) regulates the dorsoventral pattern of the Drosophila embryo (see Ferguson and Anderson, 1992 ) and the differentiation and patterning of cells in imaginal discs (Spencer et al., 1982; Posakony et al., 1991; Campbell et al., 1993, Heberlein et al., 1993). similarly, wingless (wg), a member of the wnt gene family, controls cell fates during segmentation and imaginal disc development (Morata and Lawrence, 1977; Nusslein-Volhard and Wieschaus, 1980; Baker, 1988; Martinez-Arias et al., 1988; Struhl and Basler, 1993).
A third Drosophila gene important in the specification of cell identity is hedgehog (hh) (Nusslein-Volhard and Wieschaus, 1980). hh acts with dpp and wg to control cell fate and pattern during segmentation and imaginal disc development (Hidalgo and Ingham, 1990; Ingham, 1993; Ma et al., 1993; Heberlein et al., 1993; Basler and Struhl, 1994; Heemskerk and DiNardo, 1994). hh encodes a novel protein (Lee et al., 1992; Mohler and Vani, 1992; Tabata et al., 1992; Tashiro et al., 1993) that enters the secretory pathway (Lee et al., 1992), and genetic evidence indicates the hh function is not cell autonomous (Mohler, 1988; Heberlein et al., 1993; Ma et al., 1993; Basler and Struhl, 1994), consistent with the possibility that hh acts as a signaling molecule.
The importance of hh in cell patterning in insects prompted us to search for vertebrate homologs and to examine their potential functions during early neural development. We report here the cloning of a vertebrate homolog of hh, vhh-1, from rat. Recent independent studies have identified a vertebrate homolog of hh, sonic hedgehog (shh), that is closely related to vhh-1 and appears to regulate cell patterning in the neural tube and limb bud (Echelard et al., 993; Krauss et al., 1993, Riddle et al., 1993). Here, we present evidence that vhh-1 is involved in the induction of ventral neural cell types. vhh-1 is expressed in midline structures (in particular, the node, notochord, and floor plate) at a time when these cells have inducing activity. COS cells expressing the rat vhh-1 gene induce floor plate and motor neuron differentiation in neural plate explants in vitro. Moreover, widespread expression of the rat vhh-1 gene in frog embryos leads to ectopic expression of the floor plate markers in the neural tube. These results suggest that vhh-1 expression in the notochord provides an inductive signal that is involved in the differentiation of floor plate cells, motor neurons, and possibly other cell types in the ventral neural tube.